The development of the mammalian central nervous system (CNS) begins in the early stage of fetal development and continues until the post-natal period. In the adult CNS there are primarily three cells: neurons, astrocytes and oligodendrocytes. The first stage of neural development in the embryo is cell genesis, a period of precise temporal and spatial sequence in which proliferation by stem and progenitor cells give rise to the precursors that will differentiate into mature CNS cells. The second step is a period of cell differentiation and migration where neuroblasts (cells that will give rise to neurons) and glialblasts (cells that will give rise to astrocytes and oligodendrocytes) differentiate and migrate to their final positions. The third stage of development occurs when cells acquire specific mature phenotypic qualities such as neurons expressing particular neurotransmitters. The final stage of CNS development is a period of selective cell death, wherein the death and degeneration of specific cells, fibers and connections “fine-tune” the circuitry of the nervous system.
Unlike many other tissues of the body the mammalian CNS has traditionally exhibited little capacity to generate new cells in response to injury or disease. However, the relatively recent discovery of cells within the adult CNS that exhibit stem cell characteristics in vitro (Reynolds and Weiss, 1992) together with the re-examination (Altman, 1962; Altman and Das, 1965) of small proliferative zones in the adult brain (Alvarez-Buylla et. al., 2001) have led to the belief that the adult mammalian CNS retains the ability to generate new cells and that neural stem cells are the source of the proliferating precursors.
Within the CNS, neural stem cells (NSC) can be differentiated from progenitor cells primarily on their proliferative and differentiation potential. Based on the Potten and Loeffler (Potten and Loeffler, 1990) definition, NSC can be differentiated from progenitor cells by their ability to exhibit self-maintenance, produce a large number of progeny and to produce mature cells of all three primary cells types in neural tissue.
The critical identifying feature of a stem cell is its ability to exhibit self-renewal or to generate more of itself. In its simplest definition a stem cell would be a cell with the capacity for self-maintenance. However, this definition can be problematic as a large number of cells may be considered to fulfill this criteria. A more stringent and practical definition of a stem cell, is provided by Potten and Loeffler (1990) who have defined stem cells as “undifferentiated cells capable of a) proliferation, b) self-maintenance, c) the production of a large number of differentiated functional progeny, d) regenerating the tissue after injury, and e) a flexibility in the use of these options.”
Culture systems have proven to be invaluable tools in studying and understanding the cellular and molecular properties of biological processes and systems. With respect to neural stem cells, a tissue culture method has been developed that allows the isolation, proliferation and expansion of neural stem cells and the subsequent differentiation of their progeny into the three primary cells types of the CNS (Reynolds and Weiss, 1996). Reynolds and Weiss identified a neural stem cell based on functional criteria. These criteria include the ability to 1) proliferate and generate a large number of progeny, 2) self-renew over an extended period of time in long-term cultures, and 3) continue to give rise to the primary cell types of the tissue from which they are obtained. Referred to as the Neurosphere Assay (NA) it has provided a wealth of data on the existence of neural stem cells and on their potential for therapeutic use.
Briefly, the NA involves the microdissection of embryonic through to adult CNS tissue followed by the disruption of cell to cell contacts and the generation of a suspension of single cells. Cells are plated (typically at a low density) in tissue cultureware in a defined serum-free medium in the presence of at least one proliferation-inducing growth factor (ie. Epidermal Growth Factor [EFG], basic Fibroblastic Growth Factor [bFGF] etc.). Under these conditions within 2-5 days a multipotent NSC begins to divide giving rise to a clonally derived cluster of undifferentiated cells referred to as a neurosphere (FIG. 1). In the continued presence of the proliferation inducing factor the cells in the neurosphere continues to divide resulting in an increase in the number of cells comprising the neurosphere and consequently the size of the neurosphere. Neurospheres can be collected, disrupted in to a single cell suspension, and the cells replated in culture to generate new neurospheres. Passaging of NSC in this manner results in an arithmetic increase in viable CNS precursor cells (FIG. 3).
The NA has become the standard assay for the isolation of mammalian NSC and forms the core of many assays used to understand the cellular and molecular biology of stem cells in the nervous system. For instance, it has been used to screen exogenous signaling factors for their effects on stem cell function (Shimazaki, et. al., 2001) and to help understand the in vivo biology of neural stem cells (Morshead, et. al., 1994; Alvarez-Buylla et. al., 2001). While this assay has proven valuable in advancing the field it suffers from a significant limitation.
As a population, neurospheres can be passaged at least 10 times resulting in the generation of a large number of progeny that can be subsequently differentiated into the three primary cell types found in the mammalian CNS—neurons, astrocytes and oligodendrocytes—thereby satisfying the primary requirements of a stem cell (Reynolds and Weiss, 1996). In addition, individual clonally derived spheres can be dissociated into single cells and in the presence of a mitogenic factor, new spheres are generated (self-maintenance) and the progeny can be differentiated into neurons, astrocytes and oligodendrocytes. It is currently assumed that every sphere generated in the NA is derived from a NSC. The inventors have unexpected results indicating that this is not true and that the proliferative potential of the neurospheres generated in a NA vary. Hence, while the NA identifies NSC not all neurospheres generated in this assay are derived from a NSC. Some (and maybe the majority) of the neurospheres may be neural progenitor cells with a more limited proliferative potential and possibly a different differentiation potential as well. The number of NSC determined by a NA is an overestimation and subsequent interpretations based on the results are likely to be incorrect. For instance, FIGS. 2A and 2B are representative of a population of neurospheres generated in the NA. As a hypothetical example lets say the difference between FIGS. 2A and 2B is the addition of a polypeptide growth factor called GF-X in 2B. In this case it would appear that the addition of GF-X resulted in an approximate 34% reduction in the number of neurospheres. This would be interpreted as a negative regulatory effect of GF-X on the proliferation or survival of NSC in this particular experiment. This interpretation would be correct if all neurospheres generated in the NA were derived from stem cells, however, if they are not the conclusion is invalid. An example of such an experiment can be found in U.S. Pat. No. 5,851,832 Example 43. In both of these cases change in the number of neurospheres is assumed to reflect an effect on NSC, however, unless all neurospheres generated in the NA are shown to be derived from stem cells this assumption is unfounded. Hence, a significant deficiency exists in the currently used method to study NSC.
Therefore, in view of the aforementioned deficiency attendant with prior art methods of studying NSC in vitro, a need exists in the art for an in vitro assay that can differentiate between NSC and neural progenitor cells.
A need also exists for an assay that can differentiate between different NSC based on their proliferative potential.